Cdk5/p35 functions as a crucial regulator of spatial learning and memory.

BACKGROUND: Cyclin-dependent kinase 5 (Cdk5), which is activated by binding to p35 or p39, is involved in synaptic plasticity and affects learning and memory formation. In Cdk5 knockout (KO) mice and p35 KO mice, brain development is severely impaired because neuronal migration is impaired and lamination is disrupted. To avoid these developmental confounders, we generated inducible CreER-p35 conditional (cKO) mice to study the role of Cdk5/p35 in higher brain function. RESULTS: CreER-p35 cKO mice exhibited spatial learning and memory impairments and reduced anxiety-like behavior. These phenotypes resulted from a decrease in the dendritic spine density of CA1 pyramidal neurons and defective long-term depression induction in the hippocampus. CONCLUSIONS: Taken together, our findings reveal that Cdk5/p35 regulates spatial learning and memory, implicating Cdk5/p35 as a therapeutic target in neurological disorders.

Glucose phosphorylation is required for Mycobacterium tuberculosis persistence in mice.

Mycobacterium tuberculosis (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. (13)C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt) Mtb. When both glucokinase genes, ppgK and glkA, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose in vivo and relies on glucose phosphorylation to survive during chronic mouse infections.

Both cyclin I and p35 are required for maximal survival benefit of cyclin-dependent kinase 5 in kidney podocytes.

Cyclin-dependent kinase (Cdk)-5 is activated by both cyclin I and the noncyclin activator p35 in terminally differentiated cells such as kidney podocytes and neurons. Cyclin I and p35 are restricted to podocytes in the kidney, and each limit podocyte apoptosis by activating Cdk5. To determine whether both activators are necessary, or whether they serve backup roles, a double cyclin I-p35 null mouse was generated. Experimental glomerular disease characterized by podocyte apoptosis was then induced by administering an anti-podocyte antibody. The results showed that under nonstressed conditions double mutants had normal kidney structure and function and were indistinguishable from wild-type, cyclin I(-/-), or p35(-/-) mice. In contrast, when stressed with disease, podocyte apoptosis increased fourfold compared with diseased cyclin I(-/-) or p35(-/-) mice. This resulted in a more pronounced decrease in podocyte number, proteinuria, and glomerulosclerosis. Under normal states and nephritic states, levels for the prosurvival protein Bcl-2 were lower in double cyclin I(-/-) p35(-/-) mice than the other mice. Similarly, levels of Bcl-xL, another prosurvival member, were lower in normal and nephritic double cyclin I(-/-) p35(-/-) mice but similar to single-cyclin I(-/-) mice. Moreover, levels of ERK1/2 and MEK1/2 activation were lower in nephritic double cyclin I(-/-) p35(-/-) mice but similar to single-cyclin I(-/-) mice. The results demonstrate that the activators of Cdk5, p35, and cyclin I are not required for normal kidney function. However, they play pivotal coordinated roles in maintaining podocyte survival during stress states in disease.

Sedoheptulokinase deficiency due to a 57-kb deletion in cystinosis patients causes urinary accumulation of sedoheptulose: elucidation of the CARKL gene.

The most common mutation in the nephropathic cystinosis (CTNS) gene is a homozygous 57-kb deletion that also includes an adjacent gene carbohydrate kinase-like (CARKL). The latter gene encodes a protein that is predicted to function as a carbohydrate kinase. Cystinosis patients with the common 57-kb deletion had strongly elevated urinary concentrations of sedoheptulose (28-451 mmol/mol creatinine; controls and other cystinosis patients <9) and erythritol (234-1110 mmol/mol creatinine; controls and other cystinosis patients <148). Enzyme studies performed on fibroblast homogenates derived from patients carrying the 57-kb deletion revealed 80% reduction in their sedoheptulose phosphorylating activity compared to cystinosis patients with other mutations and controls. This indicates that the CARKL-encoded protein, sedoheptulokinase (SHK), is responsible for the reaction: sedoheptulose + ATP --> sedoheptulose-7-phosphate + ADP and that deletion of CARKL causes urinary accumulation of sedoheptulose and erythritol.

Prenatal diagnosis of glycogen storage disease type 1b using denaturing high performance liquid chromatography.

Glycogen storage disease type 1b (GSD1b) is an autosomal recessive inborn error of metabolism caused by deficiency of glucose-6-phosphate translocase (G6PT1). Current laboratory diagnosis for GSD1b is established by a functional enzyme assay of glucose-6-phosphatase in both fresh and detergent-treated liver homogenates. This procedure requires liver biopsy and is impractical for routine prenatal diagnosis owing to the high morbidity of fetal liver biopsy. Recently, the gene for GSD1b has been cloned and the prevalent mutations in different ethnic groups have been determined. In this study, prenatal molecular diagnosis was performed for a Chinese family in which a previous child was born homozygous for the G149E mutation. We detected genomic sequence variants by heteroduplex formation, followed by denaturing high performance liquid chromatography (DHPLC). With this method, post-PCR analysis was shortened to 7 min. In the case we analysed, PCR products amplified from the fetal DNA yielded a single peak in the chromatogram, indicating a homozygous state in the fetus. When wild-type PCR products were mixed with fetal PCR products, two peaks were observed, indicating that the fetus was homozygous for the parental (G149E) mutation. Sequencing results confirmed this diagnosis. As a result, the pregnancy was terminated and the diagnosis was confirmed on DNA analysis of the aborted fetus. We show here that DNA mutation analysis can be used in the prenatal diagnosis of GSD1b and that DHPLC promises to be a robust technique for this and other prenatal molecular diagnoses.

H. pylori activates NF-kappaB through a signaling pathway involving IkappaB kinases, NF-kappaB-inducing kinase, TRAF2, and TRAF6 in gastric cancer cells.

BACKGROUND & AIMS: H. pylori infection on gastric epithelial cells has been shown to induce NF-kappaB activation, but the mechanism of intracellular signal conduction that leads to NF-kappaB activation is not clear. The aim of this study was to analyze the molecular mechanism responsible for H. pylori-mediated NF-kappaB activation on gastric cancer cells. METHODS: NF-kappaB activation by H. pylori was tested by using luciferase reporter assay. IkappaBalpha degradation by H. pylori infection was assessed by immunoblotting. IKKalpha and IKKbeta activation was analyzed by kinase assay. In transfection experiments, effects of dominant negative IkappaBalpha, IKKalpha, IKKbeta, NF-kappaB-inducing kinase (NIK), TRAF2, and TRAF6 mutants were investigated. The effects of an IKKbeta-specific inhibitor, aspirin, on NF-kappaB activation and IL-8 secretion were also analyzed. RESULTS: H. pylori promotes degradation of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB. In kinase assay, H. pylori induced IKKalpha and IKKbeta catalytic activity in gastric cancer cells. Transfection of kinase-deficient mutant of either IKK inhibited H. pylori-mediated NF-kappaB activation dose-dependently. Aspirin inhibited both NF-kappaB activation and IL-8 secretion induced by H. pylori. NF-kappaB activation was also inhibited by transfection of kinase-deficient NIK or a dominant negative mutant of upstream adapter protein TRAF2 or TRAF6. CONCLUSIONS: H. pylori induces NF-kappaB activation through an intracellular signaling pathway that involves IKKalpha, IKKbeta, NIK, TRAF2, and TRAF6.

Multiple transport protein defects in a patient with glycogen storage disease type 1: GSD 1b/1c beta.

A male child presented at 5 months of age with vomiting, diarrhoea, hypoglycaemia and hepatomegaly. Histology on a frozen liver biopsy suggested glycogen storage disease (GSD), while biochemical analyses confirmed an elevated glycogen content and normal activities of the GSD enzymes with the proviso that a variant of GSD 1 should be considered. The patient presented at 9 months of age with severe lactic acidosis and hypoglycaemia. A glucagon tolerance test and galactose load test on the patient produced no glycaemic response. A second biopsy was obtained and appropriately handled for the investigation of variants of the glucose-6-phosphatase enzyme (G6Pase) complex. Results showed that the patient had a deficiency of two transport proteins of the G6Pase complex, namely glucose-6-phosphate translocase and pyrophosphate translocase, i.e. GSD 1b/1c beta. These results were confirmed by additional kinetic analyses which provided confirmation of the double translocase deficiency. Evidence for inhibitors to these translocases was not found. The patient's treatment has resulted in the hypoglycaemia now being well controlled; however, at 3 years of age, height and weight are markedly lagging and he is moderately developmentally delayed. Neutropenia has not been found and neutrophil function is normal. Double enzyme deficiencies are very rare and possible explanations which might lead to this phenotype are considered. This, to the authors' knowledge, is the first report of a double translocase deficiency causing GSD type 1.

Enhanced urinary excretion of leukotriene E4 in patients with mevalonate kinase deficiency.

In patients with mevalonate kinase deficiency, urinary excretion of the leukotriene LTE4 was found to be elevated. A positive linear relationship between increased urinary excretion of mevalonate and LTE4 (n = 5) suggests that increased cysteinyl leukotriene synthesis is involved in the pathomechanisms of this disease.

Metabolic pathways for the activation of the antiviral agent 2',3'-dideoxyguanosine in human lymphoid cells.

2',3'-Dideoxyguanosine (ddGuo) is a selective inhibitor of the replication of human immunodeficiency virus in vitro and the most active antihepadnavirus nucleoside analog known in vitro and in vivo, in a Peking duck model. However, the exact route by which this and related guanosine analogs are anabolized to their putative active metabolites in target cells is controversial. The anabolic pathway for the activation of ddGuo was investigated with the use of mutant human lymphoid CCRF-CEM and WI-L2 cell lines deficient in known nucleoside kinases. Uptake of ddGuo by human lymphoid cells and subsequent conversion to mono-, di-, and triphosphorylated metabolites is dose dependent and occurs proportionately to the exogenous concentration of drug. Studies with kinase-deficient CCRF-CEM and WI-L2 mutants revealed that at least two different routes of metabolism are operating in these cells to initiate the phosphorylation of ddGuo to its active dideoxynucleotides, one being deoxycytidine (dCyd) kinase and the other a cytosolic-5'-nucleotidase acting in the anabolic direction as a phosphotransferase. The evidence for this included 1) a lower but significant accumulation of drug anabolites in dCyd kinase-deficient mutants, 2) a lack of cross-resistance of the kinase-deficient mutants to growth inhibition by ddGuo, compared with that by the related analogs dideoxycytidine and arabinosylcytosine, known substrates for dCyd kinase, and 3) identification of different phosphorylation activities for ddGuo in extracts of wild-type cells and kinase-deficient mutants. Knowledge of the enzyme systems involved in anabolism of ddGuo analogs should be important for both new drug design and optimal therapeutic application.

A selectable marker allows investigation of a nontransforming Epstein-Barr virus mutant.

The derivation of specifically mutated Epstein-Barr virus (EBV) recombinants is dependent on strategies to identify, enumerate, and clone infected B lymphocytes. In recent experiments, EBV recombinants containing a positive selection marker were identified and cloned in B-lymphoma (BL) cells infected and then plated under selective conditions (F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). We now use BL cells, for the first time, as hosts for assaying and cloning otherwise isogenic EBV recombinants carrying a hygromycin phosphotransferase (HYG) gene linked to either a nontransforming deletion mutant or a transforming wild-type EBV nuclear antigen 2 (EBNA-2) gene. Both types of recombinants converted BL cells to hygromycin resistance with similar efficiency, formed episomes, and usually expressed only EBNA-1. Only the wild-type EBNA-2 HYG gene EBV recombinant transformed primary B lymphocytes. This strategy of assaying virus on BL and primary B lymphocytes makes possible the direct assessment of the transforming efficiency of an EBV recombinant. The resultant infected BL cells are also useful for the characterization of the nontransforming recombinant EBV genomes. The HYG gene insertion in the BHLF1 open reading frame eliminated BHLF1 protein expression. The insertion and resulting BHLF1 mutation did not interfere with primary B-lymphocyte infection, growth transformation, induction of lytic infection, or virus production. Thus, these experiments also indicate that neither the BHLF1 open reading frame nor the HYG gene insertion critically affects B-lymphocyte infection in vitro.

Recent progress in the molecular genetic analysis of erythroenzymopathy.

During the relatively recent period in which normal genes for most red cell enzymes have been isolated, the techniques of molecular biology have been applied to the studies of erythroenzymopathy. Single nucleotide substitutions have been identified in aldolase, triosephosphate isomerase, glucose 6-phosphate dehydrogenase, and adenylate kinase variants by the cloning and nucleotide sequence of the patients' genes. Up to now, all of the enzyme-deficient variants which have been investigated have been caused by point mutations. An exception is a hemolytic anemia secondary to increased adenosine deaminase (ADA) activity. Red cell ADA activity increases on the order of a hundred-fold in affected individuals. The basic abnormality appears to result from overproduction of structurally normal enzyme due to abnormal transcriptional or translational efficiency.

Metabolic causes of myoglobinuria.

To evaluate the proportion of cases of myoglobinuria that can be ascribed to specific metabolic defects, we have studied eight enzymes--phosphorylase, phosphorylase kinase, phosphofructokinase (PFK), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGAM), lactate dehydrogenase (LDH), carnitine palmitoyltransferase (CPT), and myoadenylate deaminase (MAD)--in muscle biopsy specimens from 77 consecutive patients with myoglobinuria (documented in 44, suspected in 33). Enzyme defects were found in 36 patients: CPT deficiency in 17, phosphorylase deficiency in 10, phosphorylase kinase deficiency in 4, MAD deficiency in 3, PGK deficiency in 1, and a combined defect of CPT and MAD in 1. Exercise was the main precipitating factor, both in patients with and in those without detectable enzymopathies. Thirty patients had specific enzymopathies without myoglobinuria: 14 had phosphorylase deficiency, 9 had MAD deficiency, 3 had phosphorylase kinase deficiency, 3 had PFK deficiency, and 1 had PGAM deficiency. Systematic biochemical evaluation of muscle biopsy specimens revealed specific enzymopathies in about half of the patients with idiopathic myoglobinuria. The rest may have blocks of metabolic pathways not yet studied routinely, such as beta oxidation, or genetic defects of the sarcolemma, such as Becker's muscular dystrophy.

Prenatal diagnosis of I-cell disease in the first and second trimesters.

First trimester prenatal diagnosis of I-cell disease (1 case) was based on demonstration of profound deficiency of N-acetylglucosamine 1-phosphotransferase in chorionic villi and in cultured trophoblasts derived from the chorionic villus specimen. Deficiency of this enzyme in cultured amniotic fluid cells obtained via amniocentesis was the basis for prenatal diagnosis of I-cell disease in the second trimester (2 cases). In both procedures, the diagnosis was corroborated by the finding of intracellular deficiency and extracellular elevation of multiple lysosomal enzymes in the fetal cell cultures (trophoblasts and amniotic fluid cells), as well as a significant increase in several lysosomal enzyme activities in the maternal serum.

Suppression of deoxyguanosine cytotoxicity and deoxyguanosine kinase activity in mouse FM3A mammary carcinoma cell: mutants defective in hypoxanthine phosphoribosyl-transferase.

The cytotoxic effect of deoxyguanosine was examined in various cell clones isolated from cultured mouse FM3A mammary carcinoma cells. The inhibitory effect of deoxyguanosine on the growth of the wild-type cell was suppressed by the addition of hypoxanthine to the culture medium. Cell mutants defective in hypoxanthine phosphoribosyl-transferase (Hprt- mutants) were approximately 20 times less sensitive to deoxyguanosine. But the sensitivity of the Hprt+ revertants was restored and has become close to that of the wild-type cell. Both uptake and incorporation of [3H]-hypoxanthine and [3H]-deoxyguanosine did not occur in the cellular acid-soluble and insoluble fractions in Hprt- mutants but they were restored in all Hprt+ revertants. When the activities of deoxyguanosine kinase were measured immediately after preparation of the cell-free extracts from the Hprt- mutants, it was negligible but activity comparable to that of the wild-type appeared after dialysis of the extract. These results are explained by assuming accumulation of purine metabolite in Hprt- mutants which inhibited the deoxyguanosine salvage pathway.

Molecular basis of red cell enzymopathies associated with hereditary nonspherocytic hemolytic anemia.

In the past few years, very rapid advances have been made in the field of red cell enzymopathies associated with hereditary nonspherocytic hemolytic anemia, particularly in molecular basis. Nucleotide sequence and amino acid sequence of normal human red cell enzymes have been clarified in phosphofructokinase, aldolase, triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, diphosphoglycerate mutase, glucose 6-phosphate dehydrogenase, adenylate kinase and adenosine deaminase. Furthermore, in aldolase-, triosephosphate isomerase-, diphosphoglycerate mutase-, glucose 6-phosphate dehydrogenase-, and adenylate kinase deficiency, single nucleotide changes which cause single amino acid substitutions and finally hemolysis, have been found.

D-glycerate kinase deficiency as a cause of D-glyceric aciduria.

D-Glycerate kinase was measured in human livers thanks to a new, sensitive radiochemical assay. The enzyme was extremely unstable in extracts prepared in water, but was partly stabilized in a homogenization mixture containing inorganic phosphate, D-glycerate and EGTA. When extracted in such a stabilizing mixture, glycerate kinase activity amounted to 0.86 +/- 0.21 U/g in control livers and to 0.03 U/g in the liver of a patient with D-glyceric aciduria. In contrast, D-glycerate dehydrogenase (glyoxylate reductase) and triokinase activities were not deficient in the liver of the same patient. It is concluded that D-glycerate kinase deficiency is a cause of D-glyceric aciduria.

Effects of deoxycytidine and thymidine kinase deficiency on substrate cycles between deoxyribonucleosides and their 5'-phosphates.

Substrate cycles constructed from a deoxyribonucleoside kinase and a deoxyribonucleotidase contribute to the metabolism of deoxyribonucleotides in cultured cells. The two enzymes catalyze in opposite directions the irreversible interconversion between a deoxyribonucleoside and its 5'-phosphate. Depending on the balance between the two reactions the net result of the cycle's activity will be synthesis or degradation of the deoxyribonucleotide, and favor import or export of the deoxyribonucleoside. With genetically changed hamster cells (V79 and CHO) deficient in either deoxycytidine or thymidine kinase we now quantify by kinetic isotope flow experiments the contributions of the two kinases to the function of the respective cycles. For each, loss of the relevant kinase was accompanied by an increased degradation of the deoxynucleotide, a slower rate of DNA synthesis, and a longer generation time for the mutant cells. The size of the corresponding deoxyribonucleoside triphosphate pool was apparently not decreased.

The use of enzymopathic human red cells in the study of malarial parasite glucose metabolism.

The in vitro growth of Plasmodium falciparum malaria parasites was assayed in mutant red cells deficient in either diphosphoglycerate mutase (DPGM) or phosphoglycerate kinase (PGK). In addition, cDNA probes developed for human DNA sequences coding for these enzymes were used to examine the parasite genome by means of restriction endonuclease digestion and Southern blot analysis of parasite DNA. In both types of enzymopathic red cells, parasite growth was normal. In infected DPGM deficient red cells, no DPGM activity could be detected, and in normal red cells, DPGM activity declined slightly in a manner suggestive of parasite catabolism of host protein. However, in infected PGK deficient red cells, there was a 100-fold increase in PGK activity, and in normal red cells, a threefold increase in PGK activity was observed. Parasite PGK could be recovered from isolated parasites, and a marked increase in heat instability of parasite PGK as compared with the host cell enzyme was noted. Neither cDNA probe was found to cross-react with DNA sequences in the parasite genome. It is concluded that the parasite has no requirement for DPGM, and probably has no gene for this enzyme. On the other hand, the parasite does require PGK, (an adenosine triphosphate [ATP] generating enzyme) and synthesizes its own enzyme, which must have been encoded in the parasite genome. The parasite PGK gene most likely lacks sufficient homology to be detected by a human cDNA probe. Enzymopathic red cells are useful tools for elucidating the glycolytic enzymology of parasites and their co-evolution with their human hosts.

First trimester prenatal evaluation for I-cell disease by N-acetyl-glucosamine 1-phosphotransferase assay.

First trimester prenatal diagnosis was offered to a couple at risk for having a child with I-cell disease (mucolipidosis II). The prenatal evaluation was based for the first time on examination of N-acetylglucosamine 1-phosphotransferase activity, deficiency of which is the primary biochemical defect in both I-cell disease and pseudo-Hurler polydystrophy (mucolipidosis III). Heterozygote levels of this enzyme activity were determined in chorionic villi obtained at 9 weeks of gestation, as well as in cultured trophoblasts derived from this specimen, and led to the diagnosis of an unaffected fetus. This procedure has advantages over that based on detection of abnormal intracellular-extracellular distribution of lysosomal enzyme activities, which is expressed only in homozygotes and fully expressed only in cell culture specimens.